Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance

Retrovirology. 2009 Feb 11;6:14. doi: 10.1186/1742-4690-6-14.

Abstract

Background: We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT).

Methods: Recombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from Escherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments.

Results: The efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT.

Conclusion: The diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / analogs & derivatives
  • Adenine / metabolism
  • DNA Primers / genetics
  • Drug Resistance, Viral*
  • HIV Reverse Transcriptase / chemistry
  • HIV Reverse Transcriptase / genetics
  • HIV Reverse Transcriptase / metabolism*
  • HIV-1 / chemistry
  • HIV-1 / drug effects
  • HIV-1 / enzymology*
  • HIV-1 / genetics*
  • Kinetics
  • Mutation, Missense*
  • Organophosphonates / metabolism
  • RNA, Viral / genetics
  • Reverse Transcription
  • Tenofovir

Substances

  • DNA Primers
  • Organophosphonates
  • RNA, Viral
  • Tenofovir
  • reverse transcriptase, Human immunodeficiency virus 1
  • HIV Reverse Transcriptase
  • Adenine